建立一种survivin抗原肽诱导T淋巴细胞优势取用TCR Vβ基因的方法  

Establishment of a method in which the specificity TCR Vβ gene amplification was induced in survivin antigen peptide-stimulated T lymphocyte

作　　者：林燕梅[1] 沈晗[1] 邵红伟[1] 吴凤麟[1] 黄树林[1] 肖兰凤[2] 

机构地区：[1]广东药学院生命科学与生物制药学院广东省生物技术候选药物研究重点实验室,广州510006 [2]广东药学院临床医学院,广州510310

出　　处：《中国免疫学杂志》2013年第3期300-303,共4页Chinese Journal of Immunology

基　　金：国家"重大新药创制"科技重大专项(2009ZX09103-708);国家自然科学基金(31100664);广东省自然科学基金(1015102240100002)

摘　　要：目的:探讨survivin抗原肽体外诱导健康人外周血单个核细胞(PBMC)后T淋巴细胞活化情况及TCRVβ表达谱变化。方法:分离健康人PBMC经rhIL-4、rhGM-CSF诱导DCs,将成熟DCs分为阴性组(不负载肽)、未洗脱负载组(负载survivin肽)、洗脱负载组(经酸液洗脱并多聚甲醛固定后负载survivin肽),分别与自体淋巴细胞共培养诱导抗原特异性T淋巴细胞。分别于24小时、48小时、96小时和7天以Elisa法检测诱导后T淋巴细胞中IFN-γ的分泌,并于第7天通过流式细胞仪分析TCRVβ表达谱。结果:随着时间延长两实验组IFN-γ的分泌比阴性组显著增高,且洗脱负载组增加更为显著。流式细胞仪分析结果显示洗脱负载组相比于阴性组及未洗脱负载组引起TCR Vβ表达谱的改变更为明显,其中TCR亚家族Vβ7.2、Vβ12、Vβ14、Vβ16、Vβ20、Vβ22最为显著。结论:DCs经酸液洗脱表面多肽并固定的方法更能有效地诱导TCRVβ亚家族优势取用。Objective：To investigate T lymphocyte＇ s activation and TCR Vβ expression profile changes after the health human peripheral blood mononuclear cells （PBMC） were induced by survivin antigen peptide in vitro . Methods：PBMC were isolated from healthy donors and DCs was induced by IL-4, rhGM-CSF . Mature DCs were divided into 3 groups,including negative group （without loaded peptide group, NG）, loaded group without eluted and fixed（ louded peptide without the acid eluted and paraformaldehyde fixed group, LG）, and loaded group with eluted and fixed（ louded peptide with the acid eluted and paraformaldehyde fixed group,E ＋ LG）, which were co-cultured with autologous lymphocytes to induce antigen-specific T lymphocytes respectively. The IFN-γsecretion levels of induced T lymphocytes were detected by ELISA at different time-points. The changes of TCR Vβ expression were analyzed at Dayγ by flow cytometry. Results：Compared with the control group, the experimental group＇s secretion of IFN-γ increased significantly with time. Flow cytometric analysis showed that the expressions of TCR Vβ subfamily were changed significantly in E ＋ L group rather than other two groups, which included Vβ3, Vβ7.2, Vβ12, Vβ14, Vβ16, Vβ20, Vβ22 subfamilies. Conclusion：After using the acid e- luted and paraformaldehyde fixed, peptie-loaded DC can induce TCR Vβ subfamily advantage of T lymphocyte cell more effectively.

关 键 词：SURVIVIN DC负载抗原 TCR Vβ表达谱 

分 类 号：R392.9[医药卫生—免疫学]